Apical MUC1 expression revealed on the foveolar epithelium in H. pylori gastritis.

BACKGROUND: The membrane mucin MUC1 is altered in its pattern of expression in cancer, and also in other pathological situations, including Helicobacter pylori gastritis. Here we investigate the basis for the loss of apical staining of the gastric foveolar epithelium in H. pylori gastritis. METHODS: MUC1 was examined in the gastric antrum from cases of H. pylori gastritis and normal controls. We used tissue sections that were either treated or not treated with periodate to effect deglycosylation, and the monoclonal antibodies LICRLonM8, MUSE-11, CT2 and BC2. RESULTS: We show that the epitopes on the TR domain of MUC1 are partially cryptic due to glycosylation and that MUC1 is present on the apical surface of the gastric foveolar epithelium of gastritis patients. CONCLUSION: This observation suggests that there is no substantial loss of the mucin domain of MUC1 from the apical surface in gastritis, as suggested by others, but rather the H. pylori influences the glycosylation of MUC1. This paper highlights the issue of epitope specificity of monoclonal antibodies directed against disease-associated markers, specifically when they are glycoproteins, as is the case for many cancer markers.

A randomised controlled trial of ALA vs. Photofrin photodynamic therapy for high-grade dysplasia arising in Barrett's oesophagus.

Photofrin photodynamic therapy (PDT) is a licenced treatment for Barrett's oesophagus (BE) with high-grade dysplasia (HGD) but causes strictures and photosensitivity and complete reversal of dysplasia (CR-HGD) by 50 % at 5 years. 5-Aminolaevulinic acid (ALA) is an alternative treatment with non-randomised data suggesting 85 % CR-HGD and a low risk of side effects. We aimed to compare efficacy and side effect profile between the drugs. A single-centre randomised controlled trial was conducted. Presence of HGD was confirmed on three occasions by two specialist GI pathologists. Stratification was by length of BE and extent of dysplasia. Standard protocols for ALA and Photofrin-PDT were followed. Endoscopic follow-up with 2-cm four-quadrant biopsy was at 6 weeks, 4 months, and then annually. All adverse event data were collected. Sixty four patients were randomised, 34 ALA and 30 Photofrin-PDT. Median follow-up is 24 months. On intention-to-treat analysis, CR-HGD was 16/34 (47 %) with ALA-PDT and 12/30 (40 %) with Photofrin-PDT. The overall cancer incidence was 14 % (9/64). On sub-group log-rank analysis, for BE ≤ 6 cm, CR-HGD was significantly higher with ALA-PDT than Photofrin-PDT (χ(2) =5.39, p=0.02). Strictures and skin photosensitivity were significantly more common after treatment with Photofrin-PDT than ALA-PDT (33 vs. 9 % and 43 vs. 6 %, respectively, p<0.05). The rate of buried glands with either drug was significantly higher post-PDT (48 % of patients) than pre-PDT (20 %). ALA-PDT has a better risk profile than Photofrin-PDT. In patients with BE length ≤ 6 cm, preliminary results show ALA-PDT is associated with significantly higher CR-HGD. In longer segments of BE, neither PDT drug is sufficiently efficacious to warrant routine use.

Comparison of nuclear texture analysis and image cytometric DNA analysis for the assessment of dysplasia in Barrett's oesophagus.

BACKGROUND: Dysplasia is a marker of cancer risk in Barrett's oesophagus (BO), but this risk is variable and diagnosis is subject to inter-observer variability. Cancer risk in BO is increased when chromosomal instability is present. Nucleotyping (NT) is a new method that uses high-resolution digital images of nuclei to assess chromatin organisation both quantitatively and qualitatively. We aimed to evaluate NT as a marker of dysplasia in BO and compare with image cytometric DNA analysis (ICM). METHODS: In all, 120 patients with BO were studied. The non-dysplastic group (n=60) had specialised intestinal metaplasia only on two consecutive endoscopies after 51 months median follow-up (IQR=25-120 months). The dysplastic group (n=60) had high-grade dysplasia or carcinoma in situ. The two groups were then randomly assigned to a training set and a blinded test set in a 1:1 ratio. Image cytometric DNA analysis and NT was then carried out on Feulgen-stained nuclear monolayers. RESULTS: The best-fit model for NT gave a correct classification rate (CCR) for the training set of 83%. The test set was then analysed using the same textural features and yielded a CCR of 78%. Image cytometric DNA analysis alone yielded a CCR of 73%. The combination of ICM and NT yielded a CCR of 84%. CONCLUSION: Nucleotyping differentiates dysplastic and non-dysplastic BO, with a greater sensitivity than ICM. A combination score based on both techniques performed better than either test in isolation. These data demonstrate that NT/ICM on nuclear monolayers is a very promising single platform test of genomic instability, which may aid pathologists in the diagnosis of dysplasia and has potential as a biomarker in BO.

Radiofrequency ablation is effective for the treatment of high-grade dysplasia in Barrett's esophagus after failed photodynamic therapy.

Endoscopic radiofrequency ablation (RFA) is an effective treatment for high-grade dysplasia in Barrett's esophagus in ablation-naïve patients, but no studies have evaluated its use in patients in whom ablative therapy has previously failed. We describe 14 patients with residual high-grade dysplasia following aminolevulinic acid or Photofrin (porfimer sodium) photodynamic therapy (PDT). An overall complete reversal of dysplasia was achieved in 86 % with a combination of RFA and rescue endoscopic mucosal resection. The median total follow-up is 19 months. The rate of strictures was 7 % (1/14) and there was a low rate of buried glands (0.5 % follow-up biopsies). These data suggest RFA is both safe and effective for eradication of high-grade dysplasia in patients in whom PDT has failed.

Image cytometry accurately detects DNA ploidy abnormalities and predicts late relapse to high-grade dysplasia and adenocarcinoma in Barrett's oesophagus following photodynamic therapy.

BACKGROUND AND AIMS: DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barrett's oesophagus, independent of histology grade. Image cytometric DNA analysis (ICDA) is an optical technique allowing visualisation of abnormal nuclei that may be undertaken on archival tissue. Our aim was to determine the accuracy of ICDA vs FC, and evaluate DNA ploidy as a prognostic biomarker after histologically successful treatment with photodynamic therapy (PDT). METHODS: Nuclei were extracted from 40 mum sections of paraffin-embedded biopsies and processed for ICDA at UCL and FC at UW using standardised protocols. Subsequently, DNA ploidy was evaluated by ICDA on a cohort of 30 patients clear of dysplasia 1 year after aminolaevulinic acid PDT for high-grade dysplasia (HGD). The results were correlated with long-term outcome. RESULTS: In the comparative study, 93% (41 out of 44) of cases were classified identically. Errors occurred in the near-diploid region by ICDA and the tetraploid region by FC. In the cohort study, there were 13 cases of late relapse (7 cancer, 6 HGD) and 17 patients who remained free of dysplasia after a mean follow-up of 44 months. Aneuploidy post-PDT was highly predictive for recurrent HGD or cancer with a hazard ratio of 8.2 (1.8-37.8) (log-rank P=0.001). CONCLUSIONS: ICDA is accurate for the detection of DNA ploidy abnormalities when compared with FC. After histologically successful PDT, patients with residual aneuploidy are significantly more likely to develop HGD or cancer than those who become diploid. DNA ploidy by ICDA is a valuable prognostic biomarker after ablative therapy.

Gastrointestinal Kaposi's sarcoma: CD117 expression and the potential for misdiagnosis as gastrointestinal stromal tumour.

AIMS: Gastrointestinal Kaposi's sarcoma (KS) may mimic gastrointestinal stromal tumours (GISTs) histologically. Studies have shown that KS outside the gastrointestinal (GI) tract may express CD117, an antibody usually used to support a diagnosis of GIST. The aim was to evaluate the clinicopathological features of GI KS, including the expression of CD117 with and without antigen retrieval. METHODS AND RESULTS: Fourteen GI KS were assessed histologically, 12 of which were also subjected to immunohistochemistry for CD34, human herpesvirus (HHV) 8, DOG1 and CD117. CD117 immunohistochemistry was performed with and without antigen retrieval. All cases showed an infiltrative spindle cell tumour. Lamina propria infiltration, lymphoplasmacytic inflammation, extravasated red blood cells and haemosiderin were typical histological features. In all cases tumour cells were positive for CD34 and HHV8, but negative for DOG1. CD117 was positive in four of 12 cases without antigen retrieval and 10 of 12 cases with antigen retrieval. CONCLUSIONS: The microscopic distinction of GI KS from GIST can be difficult. Clues that raise the possibility of GI KS include young patient age, a history of immunosuppression, lamina propria infiltration, lymphoplasmacytic inflammation, extravasated red blood cells and haemosiderin deposition. Use of the immunomarkers CD117 (without antigen retrieval), HHV8 and DOG1 may aid in the distinction between GI KS and GIST.

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus.

OBJECTIVES: Current models of clonal expansion in human Barrett's oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett's segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett's metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. METHODS: Individual crypts across Barrett's biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. RESULTS: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett's dysplasia. CONCLUSIONS: By studying clonality at the crypt level we demonstrate that Barrett's heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett's metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands.

Molecular classification and genetic pathways in hyperplastic polyposis syndrome.

Hyperplastic Polyposis (HPPS) is a poorly characterized syndrome that increases colorectal cancer (CRC) risk. We aimed to provide a molecular classification of HPPS. We obtained 282 tumours from 32 putative HPPS patients with >or= 10 hyperplastic polyps (HPs); some patients also had adenomas and CRCs. We found no good evidence of microsatellite instability (MSI) in our samples. The epithelium of HPs was monoclonal. Somatic BRAF mutations occurred in two-thirds of our patients' HPs, and KRAS2 mutations in 10%; both mutations were more common in younger cases. The respective mutation frequencies in a set of 'sporadic' HPs were 18% and 10%. Importantly, the putative HPPS patients generally fell into two readily defined groups, one set whose polyps had BRAF mutations, and another set whose polyps had KRAS2 mutations. The most plausible explanation for this observation is that there exist different forms of inherited predisposition to HPPS, and that these determine whether polyps follow a BRAF or KRAS2 pathway. Most adenomas and CRCs from our putative HPPS patients had 'classical' morphology and few of these lesions had BRAF or KRAS2 mutations. These findings suggest that tumourigenesis in HPPS does not necessarily follow the 'serrated' pathway. Although current definitions of HPPS are sub-optimal, we suggest that diagnosis could benefit from molecular analysis. Specifically, testing BRAF and KRAS2 mutations, and perhaps MSI, in multiple polyps could help to distinguish HPPS from sporadic HPs. We propose a specific model which would have diagnosed five more of our cases as HPPS compared with the WHO clinical criteria.

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations.

Patients with multiple (5-100) colorectal adenomas (MCRAs) often have no germline mutation in known predisposition genes, but probably have a genetic origin. We collected a set of 25 MCRA patients with no detectable germline mutation in APC, MYH/MUTYH or the mismatch repair genes. Extracolonic tumours were absent in these cases. No vertical transmission of the MCRA phenotype was found. Based on the precedent of MYH-associated polyposis (MAP), we searched for a mutational signature in 241 adenomatous polyps from our MCRA cases. Somatic mutation frequencies and spectra at APC, K-ras and BRAF were, however, similar to those in sporadic colorectal adenomas. Our data suggest that the genetic pathway of tumorigenesis in the MCRA patients' tumours is very similar to the classical pathway in sporadic adenomas. In sharp contrast to MAP tumours, we did not find evidence of a specific mutational signature in any individual patient or in the overall set of MCRA cases. These results suggest that hypermutation of APC does not cause our patients' disease and strongly suggests that MAP is not a paradigm for the remaining MCRA patients. Our MCRA patients' colons showed no evidence of microadenomas, unlike in MAP and familial adenomatous polyposis (FAP). However, nuclear beta-catenin expression was significantly greater in MCRA patients' tumours than in sporadic adenomas. We suggest that, at least in some cases, the MCRA phenotype results from germline variation that acts subsequent to tumour initiation, perhaps by causing more rapid or more likely progression from microadenoma to macroadenoma.

Elastic scattering spectroscopy accurately detects high grade dysplasia and cancer in Barrett's oesophagus.

BACKGROUND AND AIMS: Endoscopic surveillance of Barrett's oesophagus currently relies on multiple random biopsies. This approach is time consuming, has a poor diagnostic yield, and significant interobserver variability. Elastic scattering spectroscopy is a real time in vivo optical technique which detects changes in the physical properties of cells. The aim of this study was to assess the potential for elastic scattering to detect high grade dysplasia or cancer within Barrett's oesophagus. METHODS: Elastic scattering spectroscopy measurements collected in vivo were matched with histological specimens taken from identical sites within Barrett's oesophagus. All biopsies were reviewed by three gastrointestinal pathologists and defined as either "low risk" (non-dysplastic or low grade dysplasia) or "high risk" (high grade dysplasia or cancer). Two different statistical approaches (leave one out and block validation) were used to validate the model. RESULTS: A total of 181 matched biopsy sites from 81 patients, where histopathological consensus was reached, were analysed. There was good pathologist agreement in differentiating high grade dysplasia and cancer from other pathology (kappa = 0.72). Elastic scattering spectroscopy detected high risk sites with 92% sensitivity and 60% specificity and differentiated high risk sites from inflammation with a sensitivity and specificity of 79%. If used to target biopsies during endoscopy, the number of low risk biopsies taken would decrease by 60% with minimal loss of accuracy. A negative spectroscopy result would exclude high grade dysplasia or cancer with an accuracy of >99.5%. CONCLUSIONS: These preliminary results show that elastic scattering spectroscopy has the potential to target conventional biopsies in Barrett's surveillance saving significant endoscopist and pathologist time with consequent financial savings. This technique now requires validation in prospective studies.

Haemorrhage into chronic plaque psoriasis as a consequence of disseminated intravascular coagulation.

We describe a patient with chronic plaque psoriasis who developed haemorrhage into pre-existing lesions during an episode of disseminated intravascular coagulation secondary to sepsis. Disseminated intravascular coagulation is a complex disorder characterized by widespread intravascular deposition of fibrin with consumption of coagulation factors and platelets and occurs as a consequence of many disorders that release procoagulant material into the circulation or cause widespread endothelial damage or platelet aggregation. As both disseminated intravascular coagulation and psoriasis occur relatively frequently in the general population we were surprised to find no previous reports of this phenomenon in the literature.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras.

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apc(min) (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apc(min), and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apc(min/+) epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apc(min/+) epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apc(min/+)-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

Helical CT pneumocolon to assess colonic tumors: radiologic-pathologic correlation.

OBJECTIVE: The objective of this study was to determine the accuracy of helical CT pneumocolon in the staging of colonic carcinomas. SUBJECTS AND METHODS: Fifty-two patients (27 men, 25 women; age range, 40-88 years old; mean age, 67 years old) with known or strongly suspected colonic disorders underwent CT pneumocolon. After bowel cleansing, administration of smooth muscle relaxant, and rectal air insufflation, CT pneumocolon was performed with 5-mm collimation, 2.5-mm reconstruction interval, and a pitch of 1.5. Two contrast administration protocols were used. Twelve patients received 100 ml of i.v. contrast material that was injected at 3 ml/sec; scan delay was 45 sec. The other 40 patients received 150 ml of contrast material at 5 ml/sec; scan delay was 25 sec. Images were prospectively evaluated. All patients had pathologic confirmation: 46 patients had resections and six patients had endoscopic biopsies. RESULTS: Diagnostic images were obtained in 47 of 52 patients. In the 47 patients, there were 38 colonic carcinomas (one synchronous), nine diverticular strictures, eight polyps, one ischemic stricture, and one normal study. Thirty of 38 carcinomas were correctly staged by CT. Sensitivity and specificity for serosal infiltration were 100% (35/35 carcinomas) and 33% (one of three carcinomas), respectively; sensitivity and specificity for lymph node involvement were 56% (nine of 16 carcinomas) and 95% (21/22 carcinomas), respectively. Four polyps that were smaller than 5 mm in diameter were not revealed by CT. Twelve of 14 benign lesions were correctly diagnosed and two were believed to be malignant. No malignant lesion was misdiagnosed. CONCLUSION: CT pneumocolon gave an overall staging accuracy of 79% in 38 carcinomas.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene.

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3' end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5' by approximately 2 kb revealing a 559-bp region showing strong homology to the proposed 5' untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of approximately 2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

The mutation rate and cancer.

The selection of advantageous mutations underlies tumorigenesis. The growth of a tumor is therefore a form of evolution at the somatic level, in which the population is comprised of individual cells within the tumor. Models of tumorigenesis have considered the relative importance of mutation and selection. We show that selection is more important than an increased mutation rate in the growth of a tumor. Some cancers may acquire a "mutator phenotype," probably leading to faster growth, but mutator phenotypes are not necessary for carcinogenesis.

Clinico-pathological features and p53 expression in left-sided sporadic colorectal cancers with and without microsatellite instability.

Defects in mismatch repair (MMR) can result in the development of a 'mutator phenotype', manifest as an increase in DNA replication errors (RERs). Patients with hereditary non-polyposis colorectal cancer (HNPCC) have germline mutations in MMR genes. These patients develop carcinomas of the colon and other specific sites at a significantly earlier age than patients with sporadic carcinomas. RERs are found in the cancers from patients with HNPCC and have been demonstrated in 10-20 per cent of sporadic colorectal cancers (CRCs). Loss of MMR may simply accelerate tumour development, but it is also possible that these tumours follow a different carcinogenetic pathway from tumours with intact MMR. In particular, it has been suggested that p53 mutations occur less often in RER-positive (RER+) sporadic colorectal cancers. In this study, the clinico-pathological features and frequency of p53 overexpression in 17 left-sided RER+ CRCs were compared with 35 left-sided RER- CRCs. No differences were found in the age and tumour stage at presentation, mucinous differentiation, or Jass prognostic grouping between these two types of CRC. Thirteen out of 17 (76 per cent) RER+ and 19/35 (54 per cent) RER- tumours showed overexpression of p53, a non-significant difference (chi 2 test). Although some previous studies have suggested differences in the clinico-pathological features and p53 expression of RER+ and RER- right-sided CRCs, our results show that these differences do not exist in left-sided cancers.

Polyclonal origin of colonic adenomas in an XO/XY patient with FAP.

It is widely accepted that tumors are monoclonal in origin, arising from a mutation or series of mutations in a single cell and its descendants. The clonal origin of colonic adenomas and uninvolved intestinal mucosa from an XO/XY mosaic individual with familial adenomatous polyposis (FAP) was examined directly by in situ hybridization with Y chromosome probes. In this patient, the crypts of the small and large intestine were clonal, but at least 76 percent of the microadenomas were polyclonal in origin.

Orofacial myiasis.

Oral myiasis is rare, particularly in western developed countries. This case report describes the presentation of oral myiasis as an enlarged submandibular lymph node associated with a buccal mass, in a Caucasian male who had recently returned from the Gambia. It highlights the need for increased clinical awareness when dealing with patients who have recently travelled overseas.

Renal cell carcinoma: comparison of morphological and flow cytometric parameters of primary tumour and invasive tumour lying within the renal vein.

Histological assessment and DNA flow cytometry have been performed on 15 kidneys containing primary adenocarcinomas which had invaded the renal vein. Comparison of morphological variables showed that samples of the intravenous tumour were more commonly composed of granular cells (53 per cent) than were samples from the main tumour mass (16 per cent), and were also of higher nuclear grade. In 7 of 14 kidneys, DNA studies showed either a higher S-phase fraction (five cases) or DNA aneuploidy (two cases) in tumour cells lying within the renal vein. The mean S-phase fraction was also shown to increase in higher nuclear grades. Thus, both morphological and biological differences exist between invasive tumour cells lying within the renal vein and those in the main tumour. This is a useful model for the investigation of venous invasion and may give a better prediction of the metastatic potential of renal cell carcinoma.